1. Field of the Invention
The present invention involves the field of ER-associated degradation (ERAD) in cells. More particularly, the present invention involves modulating of the interaction between the ubiquitin ligase gp78 and the ubiqutin conjugating enzyme Ube2G2. The present invention further involves polypepides comprising the Ube2G2 binding domain (G2BD) from gp78, nucleic acid molecules encoding G2BD, and methods of use thereof, including diagnostic and therapeutic methods, as well as screening assays.
2. Background
Cytosolic and nuclear proteins are targeted for proteasomal degradation by the addition of multiubiquitin chains. The specificity of this process is largely conferred by ubiquitin (Ub) protein ligases (E3s). E3s interact directly or indirectly with substrate and mediate transfer of Ub from Ub-conjugating enzymes (E2s) to target proteins where isopeptide linkages are formed. Two major E3 classes have been identified. Homologous to E6-AP C terminus (HECT) domain E3s accept Ub from E2, themselves forming thiol-ester intermediates with Ub. RING finger E3s bind E2 and apparently mediate the direct transfer of Ub from E2 to substrate (reviewed in Hershko, A, & Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425-479; Joazeiro, C. A. & Weissman, A. M. (2000) Cell 102, 549-552; and Weissman, A. M. (2001) Nat. Rev. Mol. Cell. Biol. 2, 169-178).
Ubiquitylation also plays essential roles in targeting of proteins for retrotranslocation and proteasomal targeting from the endoplasmic reticulum (ER) by processes collectively known as ER-associated degradation (ERAD). ERAD serves to degrade misfolded or otherwise functionally denatured proteins. Elucidation of its details has important implications for many diseases, including cystic fibrosis, neurodegenerative disorders, al antitrypsin deficiency, and tyrosinase deficiency (reviewed in Bonifacino, J. S. & Weissman, A. M. (1998) Annu. Rev. Cell Dev. Biol. 14, 19-57; Plemper, R. K. & Wolf, D. H. (1999) Trends Biochem. Sci. 24, 266-270; and Brodsky, J. L. & McCracken, A. A. (1999) Semin. Cell Dev. Biol. 10, 507-513). Additionally, ERAD has homeostatic functions in regulating hydroxymethylglutaryl-CoA reductase (Hampton, R. Y., Gardner, R. G. & Rine, J. (1996) Mol. Biol. Cell 7, 2029-2044) as well as unassembled, but otherwise apparently native, components of multisubunit cell surface receptors, such as the T cell antigen receptor (TCR) CD3-δ subunit (Yang, M., Omura, S., Bonifacino, J. S. & Weissman, A. M. (1998) J. Exp. Med. 187, 835-846). Ubiquitylation is an obligate step in ERAD that appears to be required for retrotranslocation to the cytosol and proteasomal degradation (Bordallo, J., Plemper, R. K., Finger, A. & Wolf, D. H. (1998) Mol. Biol. Cell 9, 209-222; Yu, H. & Kopito, R. R. (1999) J. Biol. Chem. 274, 36852-36858; Tiwari, S. & Weissman, A. M. (2001) J. Biol. Chem. 276, 16193-16200; Gardner, R. G., Swarbrick, G. M., Bays, N. W., Cronin, S. R., Wilhovsky, S., Seelig, L., Kim, C. & Hampton, R. Y. (2000) J. Cell Biol. 151, 69-82; and references therein). The details by which retrograde movement and proteasomal targeting occur and the means by which Ub is conjugated to sites on proteins that are not normally exposed to the cytosol remain to be fully understood (Bonifacino, J. S. & Weissman, A. M. (1998) Annu. Rev. Cell Dev. Biol. 14, 19-57; McClellan, A. J. & Frydman, J. (2001) Nat. Cell Biol. 3, E51-E53).
Much of what is known about ubiquitylation in ERAD derives from Saccharomyces cerevisiae. Two yeast E2s that associate with the ER, Ubc6p and Ubc7p, play roles in ERAD, with Ubc7p most frequently implicated (Bordallo, J., Plemper, R. K., Finger, A. & Wolf, D. H. (1998) Mol. Biol. Cell
9, 209-222; Gardner, R. G., Swarbrick, G. M., Bays, N. W., Cronin, S. R., Wilhovsky, S., Seelig, L., Kim, C. & Hampton, R. Y. (2000) J. Cell Biol. 151, 69-82; and Sommer, T. & Jentsch, S. (1993) Nature (London) 365, 176-179; and references therein). Ubc6p has a C-terminal hydrophobic anchor that localizes it to the ER membrane (Sommer, T. & Jentsch, S. (1993) Nature (London) 365, 176-179). Ubc7p has no intrinsic characteristics that predict membrane association. The recruitment of Ubc7p to the ER is instead accomplished by association with Cue1p, a small N-terminal anchored ER protein (Biederer, T., Volkwein, C. & Sommer, T. (1997) Science 278, 1806-1809). A single yeast ER resident E3 implicated in ERAD, Hrd1p or Der3p, has been identified. This E3 has the capacity to function with Ubc7p (Hampton, R. Y., Gardner, R. G. & Rine, J. (1996) Mol. Biol. Cell 7, 2029-2044; Bordallo, J., Plemper, R. K., Finger, A. & Wolf, D. H. (1998) Mol. Biol. Cell 9, 209-222; Bays, N. W., Gardner, R. G., Seelig, L. P., Joazeiro, C. A. & Hampton, P Y. (2001) Nat. Cell Biol. 3, 24-29; and Deak, P. M. & Wolf, D. H. (2001) J. Biol. Chem. 276, 10663-10669). The substrates targeted for degradation by this E3 are varied in structure, and there is little evidence of direct E3-substrate binding.
Murine orthologs of Ubc6p and Ubc7p (MmUBC6 and MmUBC7) have been characterized and are highly conserved relative to counterparts in other mammals (Tiwari, S. & Weissman, A. M. (2001) J. Biol. Chem. 276, 16193-16200; Katsanis, N. & Fisher, E. M. (1998) Genomics 51, 128-131; and Lin, H. & Wing, S. S. (1999) J. Biol. Chem. 274, 14685-14691). Of these MmUBC7, but not MrUBC6, is implicated in degradation of unassembled TCR subunits (Tiwari, S. & Weissman, A. M. (2001) J. Biol. Chem. 276, 16193-16200). No mammalian ERAD E3 analogous to yeast Hrd1p/Der3p has been characterized, nor has the existence of a mammalian Cue1p homolog been established.
gp78 was originally isolated as a membrane glycoprotein from murine melanoma cells and was implicated in cell migration (Nabi, I. R. & Raz, A. (1987) Int. J. Cancer 40, 396-402). Subsequently, gp78 was identified as the tumor autocrine motility factor receptor mediating tumor invasion and metastasis (Nabi, I. R., Watanabe, H., Silletti, S. & Raz, A. (1991) EXS 59, 163-177). The message encoding gp78 has recently been shown to be widely expressed in mouse tissues, and perusal of expressed sequence tag databases suggests that this is similarly true for both normal and diseased human tissues (Shimizu, K., Tani, M., Watanabe, H., Nagamachi, Y., Niinaka, Y., Shiroishi, T., Ohwada, S., Raz, A. & Yokota, J. (1999) FEBS Left. 456, 295-300). By using a monoclonal antibody, gp78 levels were found to be increased in a number of different human malignancies, with this correlating with metastatic potential. gp78 has been shown to be expressed on the cell surface and to exhibit colocalization with caveolin when endocytosis is arrested, with evidence for internalization and transport to the ER in a manner similar to simian virus 40 (Benlimame, N., Le, P. U. & Nabi, I. R. (1998) Mol. Biol. Cell 9, 1773-1786). Other studies suggest a substantial smooth ER distribution and association with structures that have been referred to as autocrine motility factor receptor tubules (Benlimame, N., Le, P. U. & Nabi, I. R. (1998) Mol. Biol. Cell 9, 1773-1786; Wang, H. J., Benlimame, N. & Nabi, I. (1997) J. Cell Sci. 110, 3043-3053).
Recently, the full-length cDNA for gp78 has been isolated and found to predict a 643-aa protein with at least five membrane-spanning domains (Shimizu, K., Tani, M., Watanabe, H., Nagamachi, Y., Niinaka, Y., Shiroishi, T., Ohwada, S., Raz, A. & Yokota, J. (1999) FEBS Left. 456, 295-300). Notably, the region C-terminal to the last transmembrane domain includes a RING finger consensus sequence (Shimizu, K., Tani, M., Watanabe, H., Nagamachi, Y., Niinaka, Y., Shiroishi, T., Ohwada, S., Raz, A. & Yokota, J. (1999) FEBS Left. 456, 295-300). gp78 is largely localized to the ER and has intrinsic RING finger-dependent E3 activity. In this way it can target itself and a heterologous ERAD substrate, CD3-δ, for proteasomal degradation.
Given the importance of gp78 in ERAD and in human disease, there is a need in the art for compositions and methods capable of modulating its activity.